In PROJECT III (Expressed CDH Candidate Genes Can Be Predicted, Then Functionally Validated in Animal Models and IPS Cells) we are addressing a most daunting loggerjam facing the genomic and genetics community. In addition to the challenge of priortizating the large data sets emanating from each of the genomic platforms, is to garner the resources and expertise to confirm the functionality and causality of the candidates in which a computationally significant variant is detected. In this project, each variant will be evaluated by stringent criteria that requires that the sequence variants be unreported in dbSNP and the 1000 Genomes Project, and that they are nonsynonymous and cause structural changes in the protein or introduce stop codons (Intertronic variants should affect species). Such variants will then be assessed for testing in the multiple animal model systems uniquely available In the Donahoe laboratory or through our collaborations with The Jackson Laboratory, under the direction of Carol Bult, Ph.D., the Co-PI of Project III, or the laboratory of Co-investigator Kate Ackerman, M.D., at University of Rochester, New York. If a mouse knockout is available and survives beyond El 5.5, we will breed these animals at The Jackson Laboratory and examine the diaphragms for defects equivalent to human diaphragmatic hernia. If the animals do not survive beyond El 5.5 (Right arm of Figure 2), a conditional knockout will be created using a promoter such as NKX3.2 to direct expression to the diaphragm under the direction of Dr. Ackerman. Embryos of these offspring will be examined at E15.5, for defects in the diaphragm. Finding a defect will confirm the functional importance of the expressed gene and make it a candidate in the search for downstream genes which can rescue the phenotype in high throughput screens. If a mouse knockout is not available (Middle column of Figure 2), we are equipped in the laboratory to knock down the expression of a gene of interest by direct in utero injection into RCAS receptor transgenic embryos, the RCAS-TVA vector containing the gene variant. This work will be done in the Expression Core directed by Maria Loscertales, Ph.D., who will also use the avian model to determine in ovo if there is a modification of the lung phenotype. Affected lungs can be then be cultured in organ culture and tested for rescue. Dr. Loscertales will also confirm expression by immunohistochemistry and in situ hybridization, which requires considerable expertise to be done properly in the tiny embryonic diaphragm. She also has considerable experience with micromanipulation techniques to be able to employ this technique data in the mouse embryo at E10 and in the chick in ovo. Dr. Loscertales'expertise is complemented by that of Dr. Kate Ackerman, co-investigator of Project III, who has over six years of experience in identifying the small mouse diaphragm structures and evaluating their dysmorphology.